Because rapid development of drug resistance is a significant clinical problem and new antiviral drugs are needed, we are developing antiviral drugs that target HIV-1 integrase (IN) and determining their intracellular mechanisms of action. Through collaborative studies, we have identified azido-group containing beta-diketo acid derivatives (DKAs) as potent inhibitors of HIV-1 replication. Because of a lack of strong correlation between inhibition of HIV-1 IN in vitro and antiviral activity in cell-based assays, rapid and reliable in vivo assays are needed to identify potential HIV-1 IN antagonists and characterize their intracellular activities. We are developing in vivo assays to define the intracellular mechanisms of action of DKAs. We are developing quantitative PCR assays to determine the effects of potential IN inhibitors on 2-LTR circle formation. We are also developing an in vivo DNA footprinting assay to characterize interactions between HIV-1 IN and viral DNA, and are determining the effects of potential IN inhibitors on the stability of the HIV-1 IN/viral DNA complexes. Finally, we are developing novel in vivo assays to identify new lead compounds that inhibit HIV-1 IN.